Functional characterization of the disease-associated CCL2 rs1024611G-rs13900T haplotype: The role of the RNA-binding protein HuR.

CC-chemokine ligand 2 (CCL2) is involved in the pathogenesis of several diseases associated with monocyte/macrophage recruitment, such as HIV-associated neurocognitive disorder (HAND), tuberculosis, and atherosclerosis. The rs1024611 (alleles:A>G; G is the risk allele) polymorphism in the CCL2 cis-regulatory region is associated with increased CCL2 expression in vitro and ex vivo, leukocyte mobilization in vivo, and deleterious disease outcomes. However, the molecular basis for the rs1024611-associated differential CCL2 expression remains poorly characterized. It is conceivable that genetic variant(s) in linkage disequilibrium (LD) with rs1024611 could mediate such effects. Previously, we used rs13900 (alleles:_C>T) in the CCL2 3' untranslated region (3' UTR) that is in perfect LD with rs1024611 to demonstrate allelic expression imbalance (AEI) of CCL2 in heterozygous individuals. Here we tested the hypothesis that the rs13900 could modulate CCL2 expression by altering mRNA turnover and/or translatability. The rs13900 T allele conferred greater stability to the CCL2 transcript when compared to the rs13900 C allele. The rs13900 T allele also had increased binding to Human Antigen R (HuR), an RNA-binding protein, in vitro and ex vivo. The rs13900 alleles imparted differential activity to reporter vectors and influenced the translatability of the reporter transcript. We further demonstrated a role for HuR in mediating allele-specific effects on CCL2 expression in overexpression and silencing studies. The presence of the rs1024611G-rs13900T conferred a distinct transcriptomic signature related to inflammation and immunity. Our studies suggest that the differential interactions of HuR with rs13900 could modulate CCL2 expression and explain the interindividual differences in CCL2-mediated disease susceptibility.

Exosomes induce neurogenesis of pluripotent P19 cells.

Exosomes play a role in tissue/organ development and differentiation. Retinoic acid induces differentiation of P19 cells (UD-P19) to P19 neurons (P19N) that behave like cortical neurons and express characteristic neuronal genes such as NMDA receptor subunits. Here we report P19N exosome-mediated differentiation of UD-P19 to P19N. Both UD-P19 and P19N released exosomes with characteristic exosome morphology, size, and common protein markers. P19N internalized significantly higher number of Dil-P19N exosomes as compared to UD-P19 with accumulation in the perinuclear region. Continuous exposure of UD-P19 to P19N exosomes for six days induced formation of small-sized embryoid bodies that differentiated into MAP2-/GluN2B-positive neurons recapitulating RA-induction of neurogenesis. Incubation with UD-P19 exosomes for six days did not affect UD-P19. Small RNA-seq identified enrichment of P19N exosomes with pro-neurogenic non-coding RNAs (ncRNAs) such as miR-9, let-7, MALAT1 and depleted with ncRNAs involved in maintenance of stem cell characteristics. UD-P19 exosomes were rich with ncRNAs required for maintenance of stemness. P19N exosomes provide an alternative method to genetic modifications for cellular differentiation of neurons. Our novel findings on exosomes-mediated differentiation of UD-P19 to P19 neurons provide tools to study pathways directing neuron development/differentiation and develop novel therapeutic strategies in neuroscience.

Serum carotenoids and Pediatric Metabolic Index predict insulin sensitivity in Mexican American children.

High concentrations of carotenoids are protective against cardiometabolic risk traits (CMTs) in adults and children. We recently showed in non-diabetic Mexican American (MA) children that serum α-carotene and ß-carotene are inversely correlated with obesity measures and triglycerides and positively with HDL cholesterol and that they were under strong genetic influences. Additionally, we previously described a Pediatric Metabolic Index (PMI) that helps in the identification of children who are at risk for cardiometabolic diseases. Here, we quantified serum lycopene and ß-cryptoxanthin concentrations in approximately 580 children from MA families using an ultraperformance liquid chromatography-photodiode array and determined their heritabilities and correlations with CMTs. Using response surface methodology (RSM), we determined two-way interactions of carotenoids and PMI on Matsuda insulin sensitivity index (ISI). The concentrations of lycopene and ß-cryptoxanthin were highly heritable [h2 = 0.98, P = 7 × 10-18 and h2 = 0.58, P = 1 × 10-7]. We found significant (P ≤ 0.05) negative phenotypic correlations between ß-cryptoxanthin and five CMTs: body mass index (- 0.22), waist circumference (- 0.25), triglycerides (- 0.18), fat mass (- 0.23), fasting glucose (- 0.09), and positive correlations with HDL cholesterol (0.29). In contrast, lycopene only showed a significant negative correlation with fasting glucose (- 0.08) and a positive correlation with HDL cholesterol (0.18). Importantly, we found that common genetic influences significantly contributed to the observed phenotypic correlations. RSM showed that increased serum concentrations of α- and ß-carotenoids rather than that of ß-cryptoxanthin or lycopene had maximal effects on ISI. In summary, our findings suggest that the serum carotenoids are under strong additive genetic influences and may have differential effects on susceptibility to CMTs in children.

Acute maternal oxidant exposure causes susceptibility of the fetal brain to inflammation and oxidative stress.

BACKGROUND: Maternal exposure to environmental stressors poses a risk to fetal development. Oxidative stress (OS), microglia activation, and inflammation are three tightly linked mechanisms that emerge as a causal factor of neurodevelopmental anomalies associated with prenatal ethanol exposure. Antioxidants such as glutathione (GSH) and CuZnSOD are perturbed, and their manipulation provides evidence for neuroprotection. However, the cellular and molecular effects of GSH alteration in utero on fetal microglia activation and inflammation remain elusive. METHODS: Ethanol (EtOH) (2.5 g/kg) was administered to pregnant mice at gestational days 16-17. One hour prior to ethanol treatment, N-acetylcysteine (NAC) and L-buthionine sulfoximine (BSO) were administered to modulate glutathione (GSH) content in fetal and maternal brain. Twenty-four hours following ethanol exposure, GSH content and OS in brain tissues were analyzed. Cytokines and chemokines were selected based on their association with distinctive microglia phenotype M1-like (IL-1ß, IFN γ, IL-6, CCL3, CCL4, CCL-7, CCL9,) or M2-like (TGF-ß, IL-4, IL-10, CCL2, CCL22, CXCL10, Arg1, Chi1, CCR2 and CXCR2) and measured in the brain by qRT-PCR and ELISA. In addition, Western blot and confocal microscopy techniques in conjunction with EOC13.31 cells exposed to similar ethanol-induced oxidative stress and redox conditions were used to determine the underlying mechanism of microglia activation associated with the observed phenotypic changes. RESULTS: We show that a single episode of mild to moderate OS in the last trimester of gestation causes GSH depletion, increased protein and lipid peroxidation and inflammatory responses inclined towards a M1-like microglial phenotype (IL-1ß, IFN-γ) in fetal brain tissue observed at 6-24 h post exposure. Maternal brain is resistant to many of these marked changes. Using EOC 13.31 cells, we show that GSH homeostasis in microglia is crucial to restore its anti-inflammatory state and modulate inflammation. Microglia under oxidative stress maintain a predominantly M1 activation state. Additionally, GSH depletion prevents the appearance of the M2-like phenotype, while enhancing morphological changes associated with a M1-like phenotype. This observation is also validated by an increased expression of inflammatory signatures (IL-1ß, IFN-γ, IL-6, CCL9, CXCR2). In contrast, conserving intracellular GSH concentrations eliminates OS which precludes the nuclear translocation and more importantly the phosphorylation of the NFkB p105 subunit. These cells show significantly more pronounced elongations, ramifications, and the enhanced expression of M2-like microglial phenotype markers (IL-10, IL-4, TGF-ß, CXCL10, CCL22, Chi, Arg, and CCR2). CONCLUSIONS: Taken together, our data show that maintaining GSH homeostasis is not only important for quenching OS in the developing fetal brain, but equally critical to enhance M2 like microglia phenotype, thus suppressing inflammatory responses elicited by environmental stressors.

N-Acetylcysteine in Combination with IGF-1 Enhances Neuroprotection against Proteasome Dysfunction-Induced Neurotoxicity in SH-SY5Y Cells.

Ubiquitin proteasome system (UPS) dysfunction has been implicated in the development of many neuronal disorders, including Parkinson's disease (PD). Previous studies focused on individual neuroprotective agents and their respective abilities to prevent neurotoxicity following a variety of toxic insults. However, the effects of the antioxidant N-acetylcysteine (NAC) on proteasome impairment-induced apoptosis have not been well characterized in human neuronal cells. The aim of this study was to determine whether cotreatment of NAC and insulin-like growth factor-1 (IGF-1) efficiently protected against proteasome inhibitor-induced cytotoxicity in SH-SY5Y cells. Our results demonstrate that the proteasome inhibitor, MG132, initiates poly(ADP-ribose) polymerase (PARP) cleavage, caspase 3 activation, and nuclear condensation and fragmentation. In addition, MG132 treatment leads to endoplasmic reticulum (ER) stress and autophagy-mediated cell death. All of these events can be attenuated without obvious reduction of MG132 induced protein ubiquitination by first treating the cells with NAC and IGF-1 separately or simultaneously prior to exposure to MG132. Moreover, our data demonstrated that the combination of the two proved to be significantly more effective for neuronal protection. Therefore, we conclude that the simultaneous use of growth/neurotrophic factors and a free radical scavenger may increase overall protection against UPS dysfunction-mediated cytotoxicity and neurodegeneration.

Oral delivery of curcumin bound to chitosan nanoparticles cured Plasmodium yoelii infected mice.

Curcumin has been shown to have anti malarial activity, but poor bioavailability and chemical instability has hindered its development as a drug. We have bound curcumin to chitosan nanoparticles to improve its bioavailability and chemical stability. We found that curcumin bound to chitosan nanoparticles did not degrade that rapidly in comparison to free curcumin when such particles were incubated in mouse plasma in vitro at room temperature. The uptake of bound curcumin from chitosan nanoparticles by mouse RBC was much better than from free curcumin. Oral delivery of curcumin bound chitosan nanoparticles to normal mice showed that they can cross the mucosal barrier intact and confocal microscopy detected the nanoparticles in the blood. Curcumin loaded chitosan nanoparticles when delivered orally improved the bioavailability of curcumin in the plasma and RBC. While mice infected with a lethal strain of Plasmodium yoelii (N-67) died between 8 and 9 days post infection, feeding of chitosan nanoparticles alone made them to survive for five more days. Feeding 1mg of native curcumin to infected mice per day for seven days resulted in survival of one third of mice but under the same condition when 1mg of curcumin bound to chitosan nanoparticles was fed all the mice survived. Like chloroquine, curcumin inhibited parasite lysate induced heme polymerization in vitro in a dose dependent manner and curcumin had a lower IC(50) value than chloroquine. We believe that binding of curcumin to chitosan nanoparticles increases its chemical stability and enhances its bioavailability when fed to mice. In vitro data suggest that it can inhibit hemozoin synthesis which is lethal for the parasite.

Induction of oxidative stress as a possible mechanism of the antifungal action of three phenylpropanoids.

The increasing incidence of hospital-acquired infections caused by drug-resistant pathogens, host toxicity, the poor efficacy of drugs and high treatment costs has drawn attention to the potential of natural products as antifungals in mucocutaneous infections and combinational therapies. Moreover, cellular and subcellular targets for these compounds may provide better options for the development of novel antifungal therapies. Eugenol, methyl eugenol and estragole are phenylpropanoids found in essential oil. They are known to possess pharmacological properties including antimicrobial activity. Induction of oxidative stress characterized by elevated levels of free radicals and an impaired antioxidant defence system is implicated as a possible mechanism of cell death. An insight into the mechanism of action was gained by propidium iodide cell sorting and oxidative stress response to test compounds in Candida albicans. The extent of lipid peroxidation (LPO) of cytoplasmic membranes was estimated to confirm a state of oxidative stress. Activity levels of primary defence enzymes and glutathione were thus further determined. Whereas these compounds cause fungal cell death by disrupting membrane integrity at minimum inhibitory concentrations (MIC), sub-MIC doses of these compounds significantly impair the defence system in C. albicans. The study has implications for understanding microbial cell death caused by essential oil components eliciting oxidative stress in Candida. The formation of membrane lesions by these phenylpropanoids thus appears to be the result of free radical cascade-mediated LPO.

Ocimum sanctum essential oil and its active principles exert their antifungal activity by disrupting ergosterol biosynthesis and membrane integrity.

The increasing incidence of drug-resistant pathogens and host toxicity of existing antifungals attracts attention toward the efficacy of natural products as antifungals in mucocutaneous infections and combinational therapies. The composition and antifungal activity of the essential oil obtained from Ocimum sanctum (OSEO) was studied. On GC-MS analysis, OSEO showed a high content of methyl chavicol (44.63%) and linalool (21.84%). Antifungal activity of OSEO and its two main constituents was determined against sixty clinical and five standard laboratory isolates of Candida. OSEO, methyl chavicol and linalool showed inhibitory activity toward all tested strains. The mechanism of their fungicidal action was assessed by studying their effect on the plasma membrane using flow cytometry, confocal imaging and determination of the levels of ergosterol, a fungal-specific sterol. Propidium iodide rapidly penetrated a majority of yeast cells when they were treated with OSEO concentrations just above MIC, implying that fungicidal activity resulted from extensive lesions of the plasma membrane. OSEO and its components also caused a considerable reduction in the amount of ergosterol. The present study indicates that OSEO, methyl chavicol and linalool have significant antifungal activity against Candida, including azole-resistant strains, advocating further investigation for clinical applications in the treatment of fungal infections.